Colony-stimulating factors (CSFs) are acidic glycoproteins required for the survival, proliferation and differentiation of hematopoietic progenitor cells in culture (Burgess and Metcalf, Blood 56: 947-958, 1980). Functionally, the various CSFs are defined by the type of hematopoietic colony produced in semisolid culture. Hence, granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the growth of progenitors which give rise to colonies containing granulocytes, macrophages, or a combination of both cell types (Wong et al., Science 228: 810-815, 1985). In addition to GM-CSF, granulocyte CSF (G-CSF or CSF.beta.), macrophage CSF (M-CSF or CSF-1) and multi-CSF (or IL-3) have been characterized and cloned from human sources (Souza et al., Science 232: 61-65, 1986; Kawasaki et al., Science 230: 291-296, 1985; Yang et al., Cell 47: 3-10, 1986).
The colony-stimulating factors are proteins of diverse physiologic function. Kaushansky et al. (Proc. Natl. Acad. Sci. USA 83: 3101-3105, 1986) and others (Emerson et al., J. Clin. Invest. 76: 1286-1290, 1985) have found that recombinant human GM-CSF (hGM-CSF) expressed in COS-1 cells stimulates not only neutrophilic, eosinophilic and monocyte-macrophage progenitor cells, but also megakaryocyte colony-forming cells and, in the presence of erythropoietin, erythroid and mixed erythroid-nonerythroid colony-forming cells. Further, hGM-CSF has been shown to stimulate mature neutrophils to localize at sites of inflammation (Weisbart et al., Nature 314: 361-363, 1985), mature eosinophils and monocytes to become activated and to enhance their killing of helminths (Handman and Burgess, J. Immunol. 122: 1134-1137, 1979; Vadas et al., Blood 61: 1232-1241, 1983), and mature monocytes and macrophages to enhance phagocytosis and tumor cell killing (Grabstein et al., Science 232: 506-508, 1986). In addition to these in vitro activities, recombinant hGM-CSF was recently demonstrated in primates to stimulate in vivo hematopoiesis (Donahue et al., Nature 321: 872-875, 1986).
Human GM-CSF is functionally distinguishable from human G-CSF or pluripoietin (Welte et al., Proc. Natl. Acad. Sci. USA 82: 1526-1530, 1985), human M-CSF (Kawasaki et al., Science 230: 291-296, 1985), also called CSF-1, and from human multi-CSF (Yang et al., Cell 47: 3-10, 1986), also known as IL-3. GM-CSF stimulates erythroid, eosinophilic, neutrophilic, monocytic and megakaryocytic cells, but does not stimulate mast cell colonies and is specific for human cells. In contrast, human M-CSF, a heterodimer of about 44 kDa, stimulates monocyte/macrophage colony formation almost exclusively; human G-CSF stimulates primarily the formation of neutrophilic but not eosinophilic colonies, stimulates erythroid and mixed erythroid/ non-erythroid colony formation only at a ten-folder higher concentration than is required for stimulation of granulocyte or granulocyte/macrophage colony formation, and also stimulates murine neutrophilic colony formation; and human multi-CSF stimulates mast cell colony formation.
Recombinant GM-CSF has been produced in several systems. Examples of such systems are found in the disclosures of (a) Golde et al. (U.S. Pat. No. 4,438,032), which describes the production of human GM-CSF in E. coli using a cDNA derived from the Mo cell line; (b) EP 183,350 and Grabstein et al. (ibid), which describe tile production of human GM-CSF in yeast; (c) PCT Application WO/8504188, which describes a cDNA-encoding murine GM-CSF which can be expressed in bacterial and mammalian host cells; and (d) PCT Application WO/8603225 and PCT Application 8600639, both of which describe the production of recombinant human GM-CSF in E. coli and other host cells.
Prior to the availability of recombinant human GM-CSF, many studies were conducted on the corresponding murine protein. Human and murine GM-CSFs are 60% homologous (Wong et al., ibid), but the mouse protein neither binds to nor stimulates human granulocyte or macrophage progenitor cells (Metcalf, Science 229: 16-22 1985). Murine GM-CSF has also been produced by Sparrow et al. (Proc. Natl. Acad. Sci. USA 82: 292-296, 1985) and DeLamarter et al. (EMBO J. 4: 2575-2581, 1985).
Despite the growing body of knowledge surrounding the in vitro, and now in vivo, physiology of hGM-CSF, little is known about the structural features responsible for the various functional properties of the growth factor. For example, CSFs are heavily glycosylated molecules. However, Donahue et al. (Nature 321: 872-875, 1986) studied unglycosylated GM-CSF produced in E. coli and concluded that the lack of carbohydrate had little effect on in vitro activity. In addition, Wong et al. (Cancer Cells 3: 235-241, Cold Spring Harbor Laboratory, New York, 1985) observed that natural and recombinant GM-CSFs exhibit varying molecular weights between 15 KD and 28 KD. They attributed this to differential carbohydrate addition or differential carbohydrate loss in purification, but detected no differences in specific activity or biological properties of the various fractions. Further, it has recently been reported that GM-CSF which lacks all carbohydrate failed to support erythroid progenitor cell proliferation (Burgess et al., Blood 84: 43-51, 1987).
The physiological role of the carbohydrate moieties of glycoproteins remains unclear. Ashwell and Morrell (Adv. Enzymol. Relat. Areas Mol. Biol. 41: 99-128, 1974) suggested a variety of functions for carbohydrate, including enhanced survival in the circulation, augmentation of binding to plasma proteins for transport, or enhancement of protein solubility. Hoffman et al. (J. Clin. Invest. 75: 1174-1182, 1985) found that deglycosylation of megakaryocyte colony stimulating factor by treatment with trifluoromethane sulfonate resulted in a loss of biological activity. Similarly, Sairam and Bhargavi (Science 229: 65-67, 1985) found that the carbohydrate moieties of the alpha subunit of glycoprotein hormones are involved in the transduction of the biological signal into cells.
In general, GM-CSF can be used within a variety of clinical applications where the proliferation of responsive cell types is desired. These applications include chemotherapy and bone marrow transplantation. However, in light of the uncertainty characteristic of the prior art, it would be advantageous to develop a more consistent method for producing proteins exhibiting activities similar to native human GM-CSF, and to develop such proteins which possess higher specific activity than the native molecule. The present invention fulfills this need, and further provides other related advantages.